Carbonic anhydrase IX (CA IX) is a membrane bound protein overexpressed on the surface of cancer cells in a hypoxic environment. CA IX is involved in tumor cell survival and metastasis, and increased expression correlates with poor clinical outcome, however there are no approved therapies against CA IX. Monoclonal antibodies have been used to target CA IX, but their large size limits penetration throughout a poorly vascularized tumor, and their slow blood clearance limits their use as tumor imaging agents or radiotherapeutics due to high background and toxicity. Small organic molecules that inhibit CA IX are highly non-specific, and can diffuse across cell membranes to bind to intracellular carbonic anhydrase isoforms abundant in healthy tissue. Here, we propose several strategies to engineer highly stable constrained peptides (knottins) and small molecule conjugates that selectively bind to the extracellular domain of CA IX with low nanomolar affinity. This work will identify novel CA-IX targeting peptides for clinical translation as diagnostic and therapeutic agents, and will also generate technology that could broadly be applied to target membrane receptors in cancer and other diseases. Aim 1: Develop tumor-targeting agents by engineering knottin peptides that bind to CA IX with high affinity. We will use yeast surface display to engineer knottin peptides that bind to CA IX with high affinities in the low nanomolar range. We will measure the relative binding affinities of engineered knottin peptides to CA IX expressed on the surface of tumor cells. Aim 2: Develop tumor-targeting agents by chemically conjugating small molecule CA IX inhibitors to knottin peptides. We will chemically couple small molecule CA IX inhibitors to knottin peptides to combine the CA IX targeting properties of known small molecules with the favorable tissue biodistribution afforded by knottin peptides. In addition to generating new CA IX targeting molecules, this aim will result in the development of a general technology platform to improve the biodistribution of small molecule tumor-targeting agents and will result in a novel approach for creating bi-specific tumor targeting agents. Aim 3: Measure biodistribution and tumor uptake of engineered CA IX targeting agents in living subjects. Engineered CA IX binding peptides will be tested for their ability to target hypoxic tumors in vivo. MicroPET imaging, biodistribution studies, and metabolite stability will be performed with 64Cu-labeled knottin peptides and small molecule conjugates in human tumor-bearing mouse xenograft models. This aim will further establish CA IX as a target for cancer diagnosis and therapy, and validate engineered CAIX-binding knottin peptides for additional clinical studies. PUBLIC HEALTH RELEVANCE: The availability of engineered peptides that target carbonic anhydrase IX (CA IX), a membrane bound protein overexpressed on the surface of cancer cells, will open up new research areas in tumorigenesis, cancer biology, molecular imaging, and structure-based drug design. The preclinical studies we are proposing will validate CA IX as a molecular target for cancer imaging to identify patients who would benefit most from targeted therapies and to monitor their disease progression. Moreover, this work will lay a foundation for future development of engineered CA IX-binding peptides as targeting agents for tumor-specific delivery of chemotherapeutics and radionuclides.